The toxic dinoflagellate Karenia brevis blooms annually in the Gulf of Mexico and negatively impacts human and ecosystem health through production of brevetoxins. A rapid, sensitive, specific assay for the detection of K. brevis based on nucleic acid sequence amplification has been developed and successfully adapted to a portable hand-held sensor. Our goal is to enhance, demonstrate, and transfer the portable hand-held sensor to users that monitor gulf waters.
Why We Care
Blooms of K. brevis cause widespread fish kills, and negatively affect human health when toxins become aerosolized along beaches, resulting in respiratory irritation. Brevetoxins also concentrate in shellfish during K. brevis blooms, resulting in neurotoxic shellfish poisoning if consumed.
Rapid, specific, and accurate quantification of K. brevis is required to monitor waters in shellfish harvesting areas to permit more timely and sensitive enumeration of K. brevis bloom conditions in coastal areas. Currently, an extensive phytoplankton monitoring network of volunteers and local, county, and state partners collects samples. Cells of K. brevis are manually counted and identified (in fixed or live samples) using light microscopy. Human expertise is required to discriminate K. brevis from non-toxic but morphologically similar taxa with samples processed individually. Samples are not easily enumerated at sea or in most field locations and are shipped to shore-based labs, causing delays in public access to bloom information.
What We Are Doing
A rapid, sensitive, and specific assay for the detection of K. brevis based on Nucleic Acid Sequence Based Amplification (NASBA) technology has been developed and successfully adapted for use with a hand-held, battery-operated sensor that detects isothermal amplification of nucleic acids using thermoregulated fluorometry. The hand-held sensor, coupled with the NASBA detection assay with a fluorescent probe, is dubbed the Karenia“tricorder” (likened to the fictional Star Trek life forms detector). NASBA works on RNA rather than DNA, increasing sensitivity and enabling transcript (mRNA) abundance detection, an estimate of gene expression. The assay is completed within an hour and the tricorder displays changes in signal as the assay is running allowing confirmation of cells in as little as 5–10 minutes.
Our research is broken into three specific phases:
- Enhancement of hand-held genetic tricorder sensors for K. brevis detection through research and development;
- Demonstration and validation of K. brevis tricorder sensors in field and lab trials; and
- Transfer of technology to end users and integration of genetic data into harmful algal bloom observing networks.
Phase I of the project will:
- Simplify the extraction and analysis of high-quality RNA from a variety of samples to allow field-based detection and quantification of K. brevis cells;
- Install a second fluorescence channel on the sensor to allow the addition of an internal control (K. breviscalibrator molecule) for improved quantification and detection of inhibitors;
- Increase utility of sensor through software developments (i.e., convert tricorder sensor software from laptop to mobile cell phone application).
Phase II will:
- Train end users, primarily state and county monitoring agencies, in the use of sensor (e.g., Florida Fish & Wildlife Commission Volunteer Harmful Algal Bloom Monitoring, Mote Marine Lab Beach Conditions Reporting System, Alabama Department of Public Health, Florida Department of Agriculture and Consumer Services);
- Integrate the tricorder sensor into pilot monitoring projects to allow quantitative validation with cell counts conducted by state monitoring agencies and transfer of data and technology to end users.
Phase III will:
- Transfer technology and synthesis of genetic data to end user groups;
- Integrate genetic data into existing harmful algal bloom reporting structures in the Gulf of Mexico (e.g., NOAA Harmful Algal Blooms Observing System and Gulf of Mexico Coastal Ocean Observing System).